Phosphorylated AKT preserves stallion sperm

viability and motility by inhibiting caspases 3

and 7

Juan M. Gallardo Bolanos, Carolina M. Balao da Silva, Patricia Martin Munoz, Antolin

Morillo Rodriguez, Maria Plaza Davila, Heriberto Rodriguez-Martinez, Ines M. Aparicio,

Jose A. Tapia, Cristina Ortega Ferrusola and Fernando J. Pena

Linköping University Post Print

N.B.: When citing this work, cite the original article.

Original Publication:

Juan M. Gallardo Bolanos, Carolina M. Balao da Silva, Patricia Martin Munoz, Antolin Morillo

Rodriguez, Maria Plaza Davila, Heriberto Rodriguez-Martinez, Ines M. Aparicio, Jose A.

Tapia, Cristina Ortega Ferrusola and Fernando J. Pena, Phosphorylated AKT preserves stallion

sperm viability and motility by inhibiting caspases 3 and 7, 2014, Reproduction, (148), 2, 221-

235.

http://dx.doi.org/10.1530/REP-13-0191

Copyright: BioScientifica

http://www.bioscientifica.com/

Postprint available at: Linköping University Electronic Press

http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-109219

Akt preserves sperm viability and motility in stallion sperm by inhibiting caspases 3 and 7.

1 Juan M Gallardo Bolaños, 1 Carolina M Balao da Silva, Patricia Martín Muñoz 1 Antolín Morillo

Rodríguez, 1María Plaza Dávila, 3Heriberto Rodríguez-Martínez, 2 Inés M Aparicio, 2 José A Tapia 1

Cristina Ortega Ferrusola 1 Fernando J Peña*.

1Laboratory of Equine Reproduction and Equine Spermatology. Veterinary Teaching Hospital,

2Department of Physiology Faculty of Veterinary Medicine. University of Extremadura Cáceres, Spain. 3

Department of Clinical and Experimental Medicine, Faculty of Health Sciences Linköping University,

Linköping, Sweden

*Correspondence to Dr. FJ Peña, Veterinary Teaching Hospital, Laboratory of Equine Spermatology and

Reproduction, Faculty of Veterinary Medicine University of Extremadura Avd de la Universidad s/n

10003 Cáceres Spain. E-mail fjuanpvega@unex.es

phone + 34 927-257167

fax +34 927257102

Acknowledgements

The authors received financial support for this study from Ministerio de Ciencia e Innovación-FEDER,

Madrid, Spain, grants AGL 2010-20758 (GAN), BFU2011-30261, Junta de Extremadura-FEDER (GR

10010 and PCE1002), VR- 2011-6353 and Formas-2011-512, Stockholm, Sweden.

The authors declare that there is no conflict of interest that could be perceived as prejudicing the

impartiality of the research reported

This paper is dedicated to the memory of Dr. Fernando Peña-Martín (1922-2013)

ABSTRACT

Akt, also referred to as PKB or Rac, plays a critical role in controlling survival and apoptosis. In order to

gain insight of mechanisms regulating sperm survival after ejaculation, the role of Akt was investigated in

stallion spermatozoa using a specific inhibitor and phosphoflow approach. Stallion spermatozoa were

washed and incubated in BWW media supplemented with 1% PVA in presence of 0, 10, 20, or 30M of

the Akt inhibitor SH5. The inhibitor resulted in a reduction of the percentage of sperm showing

phosphorylated Akt that reached a maximum after one hour of incubation, attributable either to

dephosphorylation or prevention of an active phosphorylation pathway. During in vitro incubation

stallion spermatozoa de-phosphorylated spontaneously, so after 4 hours of incubation treated samples did

not differ from controls. Akt inhibition decreased the proportion of motile spermatozoa (total and

progressive), and also sperm velocity. Likewise, Akt inactivation reduced membrane intactness leading to

enhanced membrane permeability, as well as a reduction in mitochondrial membrane potential

concomitant with more spermatozoa depicting active caspases 3 and 7. On the other hand, neither the

percentage of spermatozoa suffering oxidative stress, the production of mitochondrial superoxide radical,

DNA oxidation or sperm DNA fragmentation changed when Akt was inhibited. It is concluded that Akt is

needed for intactness of membrane and motility in ejaculated stallion spermatozoa, presumably acting by

inhibition of caspases 3 and 7, which would prevent progression of spermatozoa to an incomplete form of

apoptosis, for which we hereby propose the term spermptosis.

Key words: stallion, sperm, Akt, phosphoflow, pannexin, mitochondria, caspases 3 and 7

INTRODUCTION

Artificial insemination using chilled transported semen is, with most breeding registries now accepting

this reproductive technology, widely used by the equine industry (Pena FJ 2012). However, the viability

and potential fertilizing capacity of cooled stallion spermatozoa last only for a few days or, for some

individuals even only hours (Macias Garcia et al. 2012). Apoptosis, is a form of programmed cell death

executed by a family of cysteine proteases termed caspases acting via two major pathways; the intrinsic

activated by cellular stress and originating by liberation of mitochondrial pro-apoptotic proteins that

activates caspase 9. The other pathway is extrinsic, triggered by death receptors that after activation by

their ligands leads to cleavage of caspase 8 (Aitken et al. 2011). Occurrence of apoptosis in ejaculated

spermatozoa remains under debate. The theory of abortive apoptosis considers apoptosis in spermatozoa

as remnants from previous spermatogenesis (Sakkas et al. 2002), on the basis that spermatozoa are

terminal, transcriptionally silent cells. Such theory is in turn questioned by facts emanating from the

finding that biotechnological procedures such as cooling storage and cryopreservation, induce features

typical of apoptosis in ejaculated spermatozoa (Moran et al. 2008, Ortega-Ferrusola et al. 2008, Ortega

Ferrusola et al. 2009, Said et al. 2010, Aitken et al. 2012) thus suggesting that apoptotic phenomena can

be triggered after ejaculation, particularly those related to its intrinsic pathway (Pena et al. 2003b).

Unraveling whether apoptosis does indeed occur in ejaculated spermatozoa may have tremendous

implications for reproductive biotechnologies in humans or animals where sperm manipulation for ART´s

is rather extended (Pena et al. 2006, Ortega-Ferrusola et al. 2009). Akt , also referred to as PKB or Rac,

plays a critical role in controlling survival and apoptosis in somatic cells (Hers et al. 2011), and growing

evidence suggests that is also involved in the regulation of sperm motility and survival (Aparicio et al.

2005, Aquila et al. 2007, Koppers et al. 2011, Parte et al. 2012). Although the first component of this

pathway, PI3K has been the subject of several studies in sperm biology, Akt has received less attention

despite that recent research points out Akt as a key factor for sperm survival after ejaculation; acting

suppressing sperm capacitation and preventing the spermatozoa to enter in an apoptotic cascade leading

to sperm senescence and finally death (Pujianto et al. 2010). Moreover, we have recently demonstrated

that sperm death occurring during conservation of stallion spermatozoa also involves apoptotic related

phenomena, both after cryopreservation (Ortega-Ferrusola et al. 2008, Ortega Ferrusola et al. 2010) and

chilling (Macias Garcia et al. 2012). In view of these evidences and in order to determine the role of Akt

in the regulation of survival of ejaculated stallion spermatozoa, we investigated whether Akt affected

stallion sperm survival using a specific inhibitor and phosphoflow cytometry approach, that is the use of

antibodies against phosphorylated forms of a particular protein and detecting those using flow cytometry

(Tazzari et al. 2002, Krutzik et al. 2004, Schulz et al. 2007, Schulz et al. 2012). The results indicated that

survival of ejaculated spermatozoa was regulated by Akt through the inhibition of an apoptosis-like

mechanism. Such mechanism, as previously proposed in humans (Aitken et al. 2011), may relate to the

silent removal of defective spermatozoa from the female genitalia (Lessig et al. 2007). If proven similar,

such a mechanism conserved over species would make the equine spermatozoon a suitable model for

human spermatozoa, particularly in relation to studies of defectiveness and preservation, areas less

explored in human. Moreover, the utility of a phosphoflow approach in the study of sperm biology is

evidenced.

MATERIAL AND METHODS

Reagents and media

Ethidium homodimer, 5,5’,6,6’–tetrachloro-1,1’,3,3’ tetraethylbenzymidazolyl carbocianyne iodine (JC-

1), Mitrotracker deep Red, YO-PRO-1, Caspase 3 and 7 detection reagent CellEvent, Cellrox, MitoxSox

Red, Sytox Green and TUNEL assay were from Molecular Probes (Molecular Probes, Leiden The

Netherlands), Akt inhibitor SH-5, and OxyDNA assay kit were from Calbiochem (San Diego, CA), Akt

1/2 kinase inhibitor, carbenexolone and probenecid from Sigma (St Louis MO), antin pannexin-1 was

from AbCam (Cambridge UK), antiphospho Akt (pSer473), Akt (pThr 308) Alexa Flour® 488 and 647

conjugated monoclonal antibodies and IgG XP® Isotype Controls (Alexa Fluor® 488 and 647 conjugates)

were purchased from Cell Signaling Technology (Danvers, MA)

Semen collection and processing

Semen was obtained from 7 Pure Spanish horses (PRE) (three ejaculates each) individually housed at the

Veterinary Teaching Hospital of the University of Extremadura, Cáceres, Spain. Stallions were

maintained according to institutional and European regulations, and were collected on a regular basis

(two collections/week) during the 2012-breeding season. Ejaculates were collected using a pre-warmed,

lubricated Missouri model artificial vagina with an inline filter to eliminate the gel fraction. The semen

was immediately transported to the laboratory for evaluation and processing. The ejaculate was extended

1:1 in INRA-96 centrifuged (600g x 10 min) and re-suspended in BWW media supplemented with 1%

PVA to 50x106 spermatozoa/mL. All the experiments followed a split sample design with every ejaculate

divided in controls and treatment groups. Every particular experiment was repeated in three different

ejaculates from each of the seven stallions (21 replicates in total).

Detection of phosphorylated Akt (Ser 473) and Akt (Thr308) in stallion spermatozoa

Spermatozoa (1x106/mL) were washed with saline-Hepes medium and fixed in 2% paraformaldehyde in

phosphate buffer saline (PBS) at room temperature for 15 minutes. After fixation, the cells were washed

twice with PBS and once with PBS/1%BSA, permeabilized for 30 minutes using 0.1% saponin in PBS

containing 1% BSA, and incubated in the same buffer with 2 L/mL of Phospho-Akt (Ser 473) –Alexa

Fluor 488 conjugate (Cat number 4071, Cell signaling) and 2 L/mL of Phospho-Akt (Thr308) Alexa

fluor 647 conjugate catalogue number (C31E5E) for 30 minutes in the dark at 22ºC. Samples were then

washed in PBS, the pellets resuspended in 500 L of PBS and analyzed using a MACSQuant Flow

cytometer (Miltenyi Biotech, Madrid Spain) and a Bio-Rad MRC1024 confocal laser microscope with a

X60 oil-immersion objective. Hoechst 33342 was added to restrict analysis to spermatozoa and gate out

debris. The specificity of the antibodies was validated using western blotting, and negative controls

consisted of unstained samples and isotype controls, IgG XP® Isotype Controls (Alexa Fluor® 488 and

647 conjugates)

Western Blotting

Stallion semen was centrifuged and washed twice with PBS. After washing, sperm cells were sonicated

for 5 sec at 4 ºC in 100 µl of Lysis Buffer consisting in 50 mM Tris/HCl, pH 7.5, 150 mM NaCl, 1%

Triton X-100, 1% deoxycholate, 1 mM EGTA, 0.4 mM EDTA, a protease inhibitor cocktail (Complete,

EDTA-free), and 0.2 mM Na3VO4. The homogenates were clarified by centrifugation at 10,000 xg (15

min, 4°C) and the supernatant was used for analysis of protein concentration followed by dilution with 4×

SDS sample buffer. Proteins (25 µg/well) from stallion sperm lysates were fractionated by SDS-PAGE

using 4-20% polyacrylamide gradient gels and transferred to nitrocellulose membranes. After blocking,

membranes were incubated overnight at 4 °C with anti-pannexin-1 (1:1000), anti- pAkt Ser 473(1:1000),

and anti-pAkt Thr 308 (1:1000). The following day, membranes were washed twice and incubated for 45

min at 25 °C with anti-rabbit IgG -HRP conjugated secondary Ab. Membranes were then washed again,

incubated with enhanced chemiluminescence detection reagents, and, finally, exposed to Hyperfilm ECL

films (Amersham). Positive controls for pAkt Ser 473 and Thr 308 were pancreatic acini lysates, and

positive controls for Pannexin-1 were brain lysates. The intensity and molecular weight of appearing

bands were quantified using the software Scion Image for Windows, version 4.02 (Scion Corp.,

Frederick, MD), normalized to actin values.

Sperm motility

Sperm motility and kinematics were assessed using a CASA system (ISAS Proiser Valencia Spain)

(Pena et al. 2005a, Nunez-Martinez et al. 2007a, Gonzalez-Fernandez et al. 2009). Semen was loaded in a

The analysis was based on the examination of 25 consecutive digitalized images obtained from each field

using x10 negative phase contrast objective. At least three different fields were recorded to assure a

minimum of 200 spermatozoa per sample. Images were taken with a time lapse of 1 second - the image

capture speed was therefore one every 40 milliseconds. The number of objects incorrectly identified as

spermatozoa were minimized on the monitor by using the playback function. With respect to the setting

parameters for the program, spermatozoa with a VAP <15 m/s were considered immotile, while

spermatozoa with a velocity >15m/s were considered motile. Spermatozoa deviating <45 % from a

straight line were designated linearly motile and spermatozoa with a circular velocity (VCL) > 45m/s

were designated rapid sperm. Sperm motion absolute and re-calculated kinematic parameters measured

by CASA included the following: Curvilinear Velocity (VCL) m/s, Measures the sequential progression

along the true trajectory. Linear Velocity (VSL) m/s, Measures the straight trajectory of the spermatozoa

per unit time. Mean Velocity (VAP) m/s, Measures the mean trajectory of the spermatozoa per unit of

time.

Simultaneous flow cytometric assessment of subtle membrane changes, viability and oxidative stress

The following stock solutions were prepared in DMSO: Yo-Pro-1 (25 M), Ethidium Homodimer-1

(1.167 mM); and CellRox (5mM). Hoechst 33342 (1.62 mM in water ) was used to identify spermatozoa

and eliminate debris from the analysis. One mL of a sperm suspension containing 5 x 106

spermatozoa/mL was stained with 1 L of Yo-Pro-1, 1L of CellRox and 0.3L of Hoechst 33342. After

thorough mixing, the sperm suspension was incubated at RT in the dark for 25 minutes, the spermatozoa

were then washed in PBS and 0.3 L of ethidium homodimer added and incubated further 5 minutes

before reading in the flow cytometer. This staining is modified after previous protocols and distinguishes

four sperm subpopulations and the oxidative stress simultaneously (Pena et al. 2005b, Nunez-Martinez et

al. 2007b, Ortega Ferrusola et al. 2009). The first is the subpopulation of only Hoechst 33342 positive

spermatozoa, considered alive and without any membrane alteration. Another subpopulation is the Yo-

Pro-1 positive cells emitting green fluorescence. This subpopulation are those spermatozoa showing early

damage or a shift to another physiological state, since membranes become slightly permeable during the

first steps of damage, enabling Yo-Pro-1 but not ethidium homodimer to penetrate the plasma membrane.

None of these probes enters intact cells. Finally, two subpopulations of dead spermatozoa are easily

detected, either apoptotic (spermatozoa stained both with Yo-Pro-1 and ethidium homodimer, emitting

both green and red fluorescence), or necrotic spermatozoa (cells stained only with ethidium homodimer

emitting red fluorescence). Spermatozoa suffering oxidative stress emit fluorescence in the far red

spectrum, while Hoechst 33342 emit blue fluorescence. Representative cytograms of the assay are

depicted in figure 1. Positive controls for oxidative stress were samples supplemented with 800 M

SO4Fe and 200 L of H2O2 (Sigma) to induce the Fenton reaction.

Simultaneous flow cytometric detection of active caspases 3 and 7 and active mitochondria

CellEvent™ Caspase-3/7 Green Detection Reagent is a fluorogenic substrate for activated caspases 3 and

7. The reagent consists of a four amino acid peptide (DEVD) conjugated to a nucleic acid binding dye.

This cell-permeant substrate is intrinsically non‑ fluorescent, because the DEVD peptide inhibits the

ability of the dye to bind to DNA. After activation of caspase-3 and caspase-7 in apoptotic cells, the

DEVD peptide is cleaved, enabling the dye to bind to DNA and produce a bright, fluorogenic response

with an absorption/emission maxima of ~502/530 nm. One important advantage of this assay is that no

wash steps are required avoiding cell losses during washing. The following stock solutions; CellEvent

(2mM in DMSO), ethidium homodimer (1.167mM in DMSO), mitotracker deep red (0.5M in DMSO)

and Hoechst 33342 (1.62mM in water) were prepared. Spermatozoa (5x106/ml) in one ml of PBS, were

stained with 1L of cell event, 0.3L of Hoechst 33342 and 0.3L of Mitotracker deep red and incubated

in the dark at r.t. for 25 minutes, after that 0.3L of Ethidum homidimer were added and samples were

incubated further five minutes and samples were immediately run in the flow cytometer. Representative

cytograms of the assay are placed in figure 2. Since cryopreservation induces caspase activity in stallion

sperm (Ortega-Ferrusola et al. 2008) cryopreserved samples were used as positive controls (figure 2) for

caspase 3 and 7.

Evaluation of mitochondrial membrane potential (m)

The lipophilic cationic compound 5,5’,6,6’–tetrachloro-1,1’,3,3’ tetraethylbenzymidazolyl carbocianyne

iodine (JC-1) has the unique ability to differentially label mitochondria with low and high membrane

potential (Pena et al. 2003a, Ortega Ferrusola et al. 2009, Ortega-Ferrusola et al. 2009). In mitochondria

with high membrane potential, JC-1 forms multimeric aggregates emitting in the high orange wavelength

of 590 nm, when excited at 488 nm. In mitochondria with low membrane potential, JC-1 forms

monomers, that emit in the green wavelength (525 to 530 nm) when excited at 488 nm. For staining, a

3mM stock solution of JC-1 (Molecular Probes Europe, Leiden, The Netherlands) in dimetylsulfoxide

(DMSO) was prepared. From each sperm sample, 1 mL of a sperm suspension in PBS (5 x106/mL) was

stained with 0.5 µL JC-1 stock solution. The samples incubated at 37 °C in the dark for 40 minutes before

flow cytometric analysis.

Determination of DNA oxidation, 8-oxoguanine assay

This assay is based in the direct binding of a fluorescent probe to the DNA adduct 8- oxoguanine

(OxyDNA Assay Kit, Calbiochem), a major oxidation product and an important indicator of free radical-

induced DNA damage and oxidative stress. The assay was performed according to manufacturer

instructions and following previously published protocols(De Iuliis et al. 2009, Koppers et al. 2011). In

brief, spermatozoa (5 x 106/mL) were separated from each sample, washed in PBS and fixed in a 2%

paraformaldehide solution in 0,1M PBS (pH 7.6) for 15 minutes at room temperature. Cells were then

washed twice in PBS and once in PBS 1% BSA. Permeabilization was made by incubation in PBS 1%

BSA supplemented 0.1% saponin for 30 minutes. Samples were washed with 1mL of wash solution (1:25

dilution in water of Wash Concentrate provided by the manufacturer). Staining with 100μL of 1X FITC-

Conjugate (1:10 dilution of FITC-Conjugate with wash solution) was done for 60 minutes in the dark, at

room temperature. Finally, cells were washed twice and resuspended in 1 mL of PBS for flow cytometry

analysis. The amount of 8-oxiguanosine formed (excitation at 495 nm and emission at 515 nm), was

measured in the MACSQuant Analyzer 10 (Miltenyi Biotec, Bergisch Gladbach, Germany), as green

fluorescence being proportional to the oxidative damage caused to the DNA. Positive controls were made

after incubation of additional samples in 800M Fe2+ and 200M H2O2

Mitosox red assay

Generation of mitochondrial superoxide anion was investigated using Mitosox Red (MSR, Molecular

probes) as previously described (Koppers et al. 2008, Koppers et al. 2011). Stock solutions of Mitosox

Red (5mM in DMSO) and Sytox Green (0.125 mM in DMSO) were prepared. Spermatozoa (5 × 106 /mL)

were stained with a final concentration of 2 μM MiToSox Red and incubated for 15 min at 37º C,

followed by centrifugation for 5 min at 600 × g and resuspension in BWW. SYTOX Green (0.05 μM)

was then added for a final 15-min incubation. The MSR (red) and SYTOX Green (green) fluorescence

was then measured using 530/30 band pass (green) and 585/42 band pass (red) filters. Non sperm-specific

events were gated out after staining with Hoechst 33342 (0.1μL of a 16 mM solution), and10,000 cells

were examined.

TUNEL Assay Terminal TdT (deoxynucleotidyltransferase)-mediated dUTP nick end labeling assay

DNA cleavage may yield double stranded and single stranded DNA breaks. Both type of breaks can be

detected by labeling the free 3’-OH terminals with fluorescent nucleotides (labeled dUTPs) in an

enzymatic reaction catalized by TDT(Koppers et al. 2011). Samples were washed in PBS and fixed in a

2% paraformaldehide solution in 0,1M PBS (pH 7.6) for 15 minutes at room temperature. Cells were then

washed in PBS and maintained in 5mL methanol 70% at -20oC. For recognition of DNA fragmentation of

apoptotic cells, APO-BrdU TUNEL Assay Kit (Invitrogen, Molecular Probes) was used. This Kit allows

detection of 3’-hydroxyl ends, serving as starting points for terminal deoxynucleotidyl transferase (TdT),

which adds deoxyribonucleotides in a template-independent fashion. Addition of the deoxythymidine

analog 5-bromo-2’-deoxyuridine 5’-triphosphate (BrdUTP) to the TdT reaction labels the break sites.

Once incorporated into the DNA, BrdUTP is detected by an anti-BrdUTP antibody, which is labeled by

an Alexa Fluor 488 dye (Ex 495 nm, Em 519 nm), while total cellular DNA content is labeled by

propidium iodide (Ex 535 nm, Em 617 nm). Frozen cells were washed twice in wash buffer and

resuspended in the DNA-labeling solution for 60 minutes at 37oC in a water bath. Suspensions were

washed twice in rinse buffer, and the Alexa Fluor 488 dye-labeled anti-BrdU antibody was added to each

of them. Incubation was performed for 30 minutes at room temperature in the dark. Finally, 0,5mL of

propidium iodide RNase staining buffer was added, incubated for 30 minutes at room temperature in the

dark, followed by flow cytometry.

Flow Cytometry

Flow cytometric analyses were carried out with a MACSQuant Analyzer 10 (Miltenyi Biotech) flow

cytometer equipped with 3 lasers emitting at 405, 488 and 635 nm and 10 photomultiplier tubes (PTM):

V1 (Ex 405 Em 450/50) V2 (Ex 405 filter 525/50), B1 (Ex 488 filter 525/50) B2 (Ex 488 filter 585/40)

B3 (Ex 488 filter 655-730 (655LP + split 730)) B4 (Ex 499 filter 750 LP), R1 (Ex 635 filter 655-730

(655LP+split 730) and R2 (Ex 635 filter 750 LP. The system is equipped with the MACSQuantify

software. Sperm subpopulations were divided by quadrants, and the frequency of each subpopulation

quantified. Forward and sideways light scatter were recorded for a total of 50,000 events per sample.

Non-sperm events were eliminated gating the sperm population after Hoechst 33342 staining. The

instrument was calibrated daily using specific calibration beads provided by the manufacturer, and

compensation overlap performed before each specific experiment.

Statistical analysis

All experiments were repeated at least three times in independent samples (three independent ejaculates

from each of the seven stallions) and the results analyzed using ANOVA with the SPSS. 19.0 software for

Mac. Differences with a P value < 0.05 were regarded as significant

RESULTS

Identification and subcellular localization of phosphorylated AkTt (Ser 473) and Akt (Thr308) in stallion

spermatozoa.

The presence of phosphorylated Akt in stallion spermatozoa was demonstrated using specific antibodies,

western blotting, flow cytometry and confocal laser microscopy. Akt was mainly identified in the caudal

part of the post-acrosomal region and the mid-piece (Figure 3). This subcellular localization complies

with a putative role; inhibiting pro-apoptotic proteins localized in the mitochondrial membrane.

Inhibition of Akt leads to reduced stallion sperm motility and kinematics

To study the role of AKT in the maintenance of stallion sperm motility, split samples of stallion

spermatozoa were incubated in BWW media in presence of a specific inhibitor of AKT, [D-3-deoxy-2-O-

methyl-myo-inositol-1-[(R)-2- methoxy-3-(octadecyloxy) propyl hydrogen phosphate]] (SH-5)(Sethi et

al. 2008) (0 (vehicle), 10, 20 and 30M in DMSO) up to 6 hours at 37ºC in BWW media supplemented

with 1% PVA. The final concentration of DMSO in all samples was 0.01‰. This is this is a cell

permeable, reversible, and substrate competitive phosphatidylinositol analog that inhibits the activation of

Akt and select downstream substrates without decreasing phosphorylation of PDK-1 or other kinases

downstream of Ras, such as MAPK. This compound has been widely used as specific inhibitor of AKT in

a wide number of cell lines (Lan et al. 2011, Busch et al. 2012, Qiao et al. 2013, Yang et al. 2013) acting

as as a potent inducer of apoptosis, and selectively kills a variety of cancer cell lines that contain high

levels of active Akt. A second inhibitor, Akt 1/2 inhibitor was also used in additional experiment (Gilot

et al. 2010). The Akt phosphorylation status was monitored using flow cytometry (Fig 4). In stallion

spermatozoa Akt appeared phosphorylated at Thr308 and Ser473. Inhibition of Akt phosphorylation was

monitored after 1 and four hours of incubation. Time of incubation had a significant effect on Akt

phosphorylation, with the percentage of spermatozoa with phosphorylated Akt both at Thr 308 and Ser

473 decreasing significantly after four hours of incubation (Figure 4). After one hour of incubation in

presence of SH-5 the percentage of spermatozoa depicting phosphorylated Akt decreased significantly,

however after 4 hours of incubation differences among control and treated samples were no longer

different (Figure 4), the same effect was observed using Akt 1/2 inhibitor (Fig 9 D). After one, two, four

or six hours of incubation, sperm motility and velocities were determined using CASA analysis. After one

hour of incubation, both Akt inhibitors induced, at 30M, a significant decrease in the percentage of total

motile spermatozoa (30% vs 50% in controls), progressive sperm (6% vs 18% in controls), circular (39. 5

vs 63.5 m/s in controls), average (21.5 vs 36.7 m/s in controls) and straight line velocities (12.5 vs

21m/s in controls) (p<0.05). After two hours of incubation at 37º C, the inhibitors significantly reduced

circular velocity and average velocity (p<0.05) (figure 5 and figure 9 A and B). These data suggest that

AKT plays a role in the regulation of stallion sperm motility and velocities. After 4 and 6 hours of

incubation all parameters of sperm motility and kinematics were reduced (p<0.01), independently of

treatment.

Inhibition of Akt increases sperm membrane permeability and leads to sperm death

In order to determine whether Akt relates to early changes in membrane integrity and permeability,

stallion spermatozoa were analyzed using flow cytometry after incubation in presence or absence of the

Akt inhibitor in BWW media up to 6 hours at 37ºC. After one hour of incubation at 37ºC, the percentage

of spermatozoa with intact membranes decreased significantly (p<0.05) from 50% in controls to 38% in

presence of 30M Akt inhibitor. Simultaneously, the percentage of spermatozoa with increased

membrane permeability, apoptotic and of necrotic spermatozoa increased (P<0.05)(figure 6). However,

the effect of the inhibitor disappeared after longer incubation periods, although the percentage of dead

spermatozoa was higher after 6 hours of incubation at 37 ºC in presence of 30M Akt inhibitor.

Pannexin-1 channel is present in stallion spermatozoa, and YoPro-1 uses this channel to penetrate the

sperm membrane

In somatic cells YoPro-1 detects early apoptotic cells, since this probe uses specific channels (Pannexin-

1) to penetrate the cell membrane; these channels open in the very early stages of apoptosis (Elliott et al.

2009, Chekeni et al. 2010, Sandilos et al. 2012). In order to confirm the presence of this channel in sperm

and that YoPro-1 uses the pannexin-1 channel to penetrate the sperm membrane a specific experiment

was conducted. . First the presence of Pannexin-1 channels was investigated using Western Blotting and

specific antibodies, and secondly the channel was blocked using two unrelated inhibitors (probenecid

1mM an carbenexolone 100m) and sperm was stained with YoPro-1. Pannexin-1 was present in stallion

spermatozoa, moreover Probenecid and carbenexolone significantly reduced the percentage of YoPro-1+

(Figure 7) spermatozoa supporting that YoPro-1 uptake in this population of spermatozoa is due to the

opening of pannexin-1 channels. This may also indicate that these spermatozoa are in a early stage of an

apoptotic phenomena similar to the situation described in somatic cells (Chekeni et al. 2010) .

Inhibition of Akt phosphorylation (Ser 473) and (Thr308) increases caspase 3 and 7 activity in stallion

spermatozoa

One possible mechanism to explain our findings is that the phosphorylation status of Akt participates in

the regulation of an apoptotic-like mechanism. To test this hypothesis, we evaluated caspase 3 and 7

activation in stallion spermatozoa after incubation in presence of the Akt inhibitor. If Akt is inhibiting an

apoptotic mechanism the inhibition of its activity shall lead to an increase in caspase 3 and 7 activities.

After one hour of incubation, all concentrations tested reduced the percentage of caspase-negative, live

spermatozoa in a positive dose-dependent manner (p<0.01), (figure 8). The percentage of caspase 3

positive spermatozoa also increased in a dose dependent manner, being this effect especially evident after

four hours of incubation at 37º C (figure 8 C). These results indicate that Akt phosphorylation at Ser 473

and Thr308 inhibits caspase 3and 7 activation in ejaculated stallion spermatozoa. In order to provide

further evidences supporting that these effects were due to Akt inhibition a further experiment was

conducted using simultaneously two different Akt inhibitors (SH-5 and Akt 1-2 Kinase inhibitor 0 and

30μM) and addressing its effects on Akt phosphorylation, sperm motility and velocity and caspase 3 and

7 activation after 1 hour of incubation at 37ºC. Both inhibitors showed similar effects, with a reduction in

sperm motilities and velocities, increases in caspase 3 and 7 activities and in dephosphorylation of Akt

(figure 9).

Inhibition of Akt reduces mitochondrial membrane potential in stallion spermatozoa

To study the effect of the inhibition of Akt phosphorylation on sperm mitochondria two different assays

were performed. Mitochondrial membrane potential was studied after JC-1 and Mitotracker deep red

staining. Inhibition of Akt phosphorylation affected, in a dose and time dependent manner, mitochondrial

membrane potential of stallion spermatozoa. After one hour of incubation, 30M of Akt inhibitor

significantly reduced mitochondrial membrane potential, evaluated with two probes (figures 10 and 11).

After two hours of incubation, 20 and 30 M of Akt inhibitor decreased mitochondrial membrane

potential when determined with mitotracker deep red (figure 10), but not with JC-1. For the latter,

mitochondrial membrane potential was only significantly decreased at 30 M (figure 11).

Inhibition of Akt does not increase the production of reactive oxygen species (ROS), mitochondrial

superoxide production and DNA oxidation, and does not increase DNA fragmentation

Since disruption of mitochondria may lead to increased ROS-production and oxidative stress (Koppers et

al. 2008), the effect of an Akt inhibitor was tested. However, the differences observed in the production

of ROS were not statistically significant (figure 12). Neither was there an increase in mitochondrial

superoxide production when Akt phosphorylation was inhibited (data not shown). Moreover, inhibition of

Akt phosphorylation did not lead to significant increases in DNA oxidation on stallion spermatozoa (data

not shown). Since DNA fragmentation is a landmark of apoptosis, a TUNEL assay was used, but

inhibition of Akt had no significant effect on the percentage of fragmented DNA (data not shown).

DISCUSSION

The presence and role of Akt in stallion spermatozoa was investigated using specific inhibitors. The

overall results indicated that Akt maintains sperm survival after ejaculation through the inhibition of

caspase 3 and 7. The PI3 kinase/Akt pathway has been previously studied in spermatozoa using specific

inhibitors such as Wortmannin and LY294002, but to the authors´ knowledge this is the first time that a

specific inhibitors of Akt are used to study the Akt pathway in mammalian spermatozoa, and reveals a

direct effect of Akt inhibiting caspase 3 and 7, since Akt inhibition rapidly lead to caspase 3 and 7

activation. The data thus suggest that inhibition of an apoptotic like mechanism, probably sperm specific,

plays a role in the survival of ejaculated stallion spermatozoa, and confirm similar recent findings in

humans (Koppers et al. 2011) supporting the idea that post ejaculatory survival of stallion and human

spermatozoa is tightly regulated through the inhibition of an truncated apoptotic cascade. This inhibition

is lost over the incubation time, since spontaneous dephosphorylation of Akt occurs, suggesting that this

is a mechanism to remove redundant spermatozoa in the female genital tract (Aitken et al. 2011). Our

study suggest that Akt is necessary to maintain sperm motility, viability and mitochondrial membrane

potential through an inhibition of the activation of caspases 3 and 7. This was hereby demonstrated by the

rapid increase in caspase 3 and 7 activity upon inhibition of Akt. Inhibition of Akt was accompanied by

decreases in the percentage of sperm motility and velocity, increases in sperm membrane permeability

and decreases of mitochondrial membrane potential, all suggesting a role for Akt in maintaining sperm

viability. Although we show evidence for each point, the link between them remains to be determined.

These findings strongly suggest that stallion sperm death during storage is largely an apoptotic

phenomena, as has been previously proposed (Ball 2008, Ortega-Ferrusola et al. 2008, da Silva et al.

2011, Macias Garcia et al. 2012). Other forms of sperm dead during storage are also possible, and after 6

hours of storage we evidenced and increase in the percentage of necrotic sperm incubated in presence of

30 M SH-5, however most of these population are also caspase +, suggesting an apoptotic phenomena

behind, moreover when a different Akt inhibitor was used the same effect was reproduced further

suggesting that Akt inhibition leads to a truncated apoptotic pathway leading to cessation of sperm

motility and death. Although we present strong evidences of the existence of this pathway remains to be

fully described; for example recent evidences suggest that a extrinsic, mediated by death receptors,

pathway may also be present in spermatozoa (Macias Garcia et al. 2012, Mendoza et al. 2013)

Moreover we demonstrated for the first time the presence of Pannexin-1 channel in stallion spermatozoa,

and a putative role in early stages of membrane destabilization is proposed as evidenced in somatic cells

(Elliott et al. 2009, Chekeni et al. 2010); in addition, since YoPro-1 uptake occurs through pannexin-1

channels, the value of the YoPro-1 assay in the detection of this early stages of apoptosis was evidenced.

Recently we demonstrated a high and negative correlation between YoPro+ spermatozoa and ATP

content in stallion spermatozoa (Balao da Silva et al. 2013), further supporting this theory, since ATP

depletion in early apoptotic cells occur through pannexin-1 channels as well (Elliott et al. 2009)

Apoptotic mechanisms are involved in cryodamage in mammalian spermatozoa (Ortega Ferrusola et al.

2010, Said et al. 2010) and recently anti-apoptotic proteins have been successfully used to improve sperm

survival after thawing (Shimokawa et al. 2012). In our experiment, Akt inhibition was not accompanied

with increased ROS production and DNA fragmentation, contrary to findings in humans where inhibition

of PI3K was accompanied with increased mitochondrial production of reactive oxygen species and

oxidative DNA damage, but without changes in mitochondrial membrane potential (Koppers et al. 2011).

This discrepancy could be attributed to species differences and/or to the differential effect of the inhibitor

used in the PI3K/AKT pathway, since we inhibited specifically Akt, that is located downstream of PI3K.

Inhibition of PI3K may also affect other downstream targets. In relation to this, inhibition of PI3K results

in an increase in cAMP levels and in the tyrosine phosphorylation of PKA-anchoring protein AKAP3 in

human spermatozoa, suggesting a negative role for PI3-K in the regulation of motility (Luconi et al.

2001, du Plessis et al. 2004, Luconi et al. 2005). Moreover, studies in pig spermatozoa showed no effect

of PI3K on the percentage of motile or progressive motile spermatozoa, but evidenced an increase in

sperm velocity (Aparicio et al. 2005). In addition, both Wortmannin and LY294002 lack specificity and

provoke different responses on spermatozoa (Nauc et al. 2004). A recent study(Pujianto et al. 2010)

showed more intense changes in caspase activation and motility decreases after incubation of human

spermatozoa in presence of an Akt inhibitor than in presence of the PI3K inhibitor Wortmannin. In

contrast to human sperm, Akt inhibition in stallion spermatozoa decreased mitochondrial membrane

potential but did not lead to oxidative stress, thus supporting the existence of species-specific features in

the apoptotic mechanisms in sperm senescence. Previous reports from our laboratory indicate that the

mitochondrial permeability transition pore has a role in the apoptotic mechanism associated to sperm

senescence induced by cryopreservation (Ortega Ferrusola et al. 2010); agreeing with the role of Akt

preventing translocation of Bax from the cytosol to the mitochondria.

Inhibition of Akt did not result in significant increases of DNA fragmentation, suggesting that this

apoptotic mechanism does not lead to DNA damage, as has previously been suggested in human

spermatozoa (Aitken & Koppers 2011, Koppers et al. 2011, Aitken et al. 2012). This has been attributed

to the special architecture of the spermatozoa that prevents endonucleases activated during the apoptotic

process at the mid-piece, to reach the sperm nucleus (Koppers et al. 2011). Another possible explanation

arises due to the relatively high resistance of stallion spermatozoa to oxidative insults when compared to

those in human (Ortega Ferrusola et al. 2009), a factor that probably relates to the different lipid

composition of the sperm membranes between species (Garcia et al. 2011, Macias Garcia et al. 2011).

Multicolor flow cytometry for the study of sperm biology is worth mentioning, alongside the

development of new assays for the simultaneous evaluation of several sperm characteristics and to

improve the discrimination from debris in the analysis, a major concern in many flow cytometric sperm

assays (Petrunkina & Harrison 2010, Petrunkina & Harrison 2011b, Petrunkina & Harrison 2011a). In

short, inhibition of a sperm-specific apoptotic mechanism is involved in the survival of stallion

spermatozoa after ejaculation; this special form of apoptosis in spermatozoa could well be termed

spermptosis. Regulation of sperm survival after ejaculation emerges as an interesting area of research

with relevant implications for the study of infertility in humans, particularly for the lack of studies dealing

with intrinsic sperm dysfunction, and developments of reproductive technologies in animals. In addition,

stallion spermatozoa experiences an apoptosis phenomenon leading to cellular senescence after

ejaculation sharing biochemical and molecular characteristics with human sperm, thus emerging as a

suitable model for the study of apoptotic mechanisms involved in male factor human infertility

REFERENCES

Aitken RJ, De Iuliis GN, Gibb Z & Baker MA 2012 The simmet lecture: new horizons on an old landscape--oxidative stress, DNA damage and apoptosis in the male germ line. Reproduction in domestic animals = Zuchthygiene 47 Suppl 4 7-14.

Aitken RJ, Findlay JK, Hutt KJ & Kerr JB 2011 Apoptosis in the germ line. Reproduction 141 139-150.

Aitken RJ & Koppers AJ 2011 Apoptosis and DNA damage in human spermatozoa. Asian journal of andrology 13 36-42.

Aparicio IM, Gil MC, Garcia-Herreros M, Pena FJ & Garcia-Marin LJ 2005 Inhibition of phosphatidylinositol 3-kinase modifies boar sperm motion parameters. Reproduction 129 283-289.

Aquila S, Middea E, Catalano S, Marsico S, Lanzino M, Casaburi I, Barone I, Bruno R, Zupo S & Ando S 2007 Human sperm express a functional androgen receptor: effects on PI3K/AKT pathway. Human reproduction 22 2594-2605.

Balao da Silva CM, Ortega Ferrusola C, Morillo Rodriguez A, Gallardo Bolanos JM, Plaza Davila M, Morrell JM, Rodriguez Martinez H, Tapia JA, Aparicio IM & Pena FJ 2013 Sex sorting increases the permeability of the membrane of stallion spermatozoa. Animal reproduction science 138 241-251.

Ball BA 2008 Oxidative stress, osmotic stress and apoptosis: impacts on sperm function and preservation in the horse. Animal reproduction science 107 257-267.

Busch F, Mobasheri A, Shayan P, Lueders C, Stahlmann R & Shakibaei M 2012 Resveratrol modulates interleukin-1beta-induced phosphatidylinositol 3-kinase and nuclear factor kappaB signaling pathways in human tenocytes. The Journal of biological chemistry 287 38050-38063.

Chekeni FB, Elliott MR, Sandilos JK, Walk SF, Kinchen JM, Lazarowski ER, Armstrong AJ, Penuela S, Laird DW, Salvesen GS, Isakson BE, Bayliss DA & Ravichandran KS 2010 Pannexin 1 channels mediate 'find-me' signal release and membrane permeability during apoptosis. Nature 467 863-867.

da Silva CM, Macias-Garcia B, Miro-Moran A, Gonzalez-Fernandez L, Morillo-Rodriguez A, Ortega-Ferrusola C, Gallardo-Bolanos JM, Stilwell G, Tapia JA & Pena FJ 2011 Melatonin reduces lipid peroxidation and apoptotic-like changes in stallion spermatozoa. Journal of pineal research 51 172-179.

De Iuliis GN, Thomson LK, Mitchell LA, Finnie JM, Koppers AJ, Hedges A, Nixon B & Aitken RJ 2009 DNA damage in human spermatozoa is highly correlated with the efficiency of chromatin remodeling and the formation of 8-hydroxy-2'-deoxyguanosine, a marker of oxidative stress. Biology of reproduction 81 517-524.

du Plessis SS, Franken DR, Baldi E & Luconi M 2004 Phosphatidylinositol 3-kinase inhibition enhances human sperm motility and sperm-zona pellucida binding. International journal of andrology 27 19-26.

Elliott MR, Chekeni FB, Trampont PC, Lazarowski ER, Kadl A, Walk SF, Park D, Woodson RI, Ostankovich M, Sharma P, Lysiak JJ, Harden TK, Leitinger N & Ravichandran KS 2009 Nucleotides released by apoptotic cells act as a find-me signal to promote phagocytic clearance. Nature 461 282-286.

Garcia BM, Fernandez LG, Ferrusola CO, Salazar-Sandoval C, Rodriguez AM, Martinez HR, Tapia JA, Morcuende D & Pena FJ 2011 Membrane lipids of the stallion spermatozoon in relation to sperm quality and susceptibility to lipid peroxidation. Reproduction in domestic animals = Zuchthygiene 46 141-148.

Gilot D, Giudicelli F, Lagadic-Gossmann D & Fardel O 2010 Akti-1/2, an allosteric inhibitor of Akt 1 and 2, efficiently inhibits CaMKIalpha activity and aryl hydrocarbon receptor pathway. Chemico-biological interactions 188 546-552.

Gonzalez-Fernandez L, Ortega-Ferrusola C, Macias-Garcia B, Salido GM, Pena FJ & Tapia JA 2009 Identification of protein tyrosine phosphatases and dual-specificity phosphatases in mammalian spermatozoa and their role in sperm motility and protein tyrosine phosphorylation. Biology of reproduction 80 1239-1252.

Hers I, Vincent EE & Tavare JM 2011 Akt signalling in health and disease. Cellular signalling 23 1515-1527.

Koppers AJ, De Iuliis GN, Finnie JM, McLaughlin EA & Aitken RJ 2008 Significance of mitochondrial reactive oxygen species in the generation of oxidative stress in spermatozoa. The Journal of clinical endocrinology and metabolism 93 3199-3207.

Koppers AJ, Mitchell LA, Wang P, Lin M & Aitken RJ 2011 Phosphoinositide 3-kinase signalling pathway involvement in a truncated apoptotic cascade associated with motility loss and oxidative DNA damage in human spermatozoa. The Biochemical journal 436 687-698.

Krutzik PO, Irish JM, Nolan GP & Perez OD 2004 Analysis of protein phosphorylation and cellular signaling events by flow cytometry: techniques and clinical applications. Clinical immunology 110 206-221.

Lan TH, Xu ZW, Wang Z, Wu YL, Wu WK & Tan HM 2011 Ginsenoside Rb1 prevents homocysteine-induced endothelial dysfunction via PI3K/Akt activation and PKC inhibition. Biochemical pharmacology 82 148-155.

Lessig J, Spalteholz H, Reibetanz U, Salavei P, Fischlechner M, Glander HJ & Arnhold J 2007 Myeloperoxidase binds to non-vital spermatozoa on

phosphatidylserine epitopes. Apoptosis : an international journal on programmed cell death 12 1803-1812.

Luconi M, Marra F, Gandini L, Filimberti E, Lenzi A, Forti G & Baldi E 2001 Phosphatidylinositol 3-kinase inhibition enhances human sperm motility. Human reproduction 16 1931-1937.

Luconi M, Torcia S, Grillo D, Fiorenza MT, Forti G, Mangia F & Baldi E 2005 Enhancement of mouse sperm motility by the PI3-kinase inhibitor LY294002 does not result in toxic effects on preimplantation embryo development. Human reproduction 20 3500-3504.

Macias Garcia B, Gonzalez Fernandez L, Ortega Ferrusola C, Morillo Rodriguez A, Gallardo Bolanos JM, Rodriguez Martinez H, Tapia JA, Morcuende D & Pena FJ 2011 Fatty acids and plasmalogens of the phospholipids of the sperm membranes and their relation with the post-thaw quality of stallion spermatozoa. Theriogenology 75 811-818.

Macias Garcia B, Ortega Ferrusola C, Aparicio IM, Miro-Moran A, Morillo Rodriguez A, Gallardo Bolanos JM, Gonzalez Fernandez L, Balao da Silva CM, Rodriguez Martinez H, Tapia JA & Pena FJ 2012 Toxicity of glycerol for the stallion spermatozoa: effects on membrane integrity and cytoskeleton, lipid peroxidation and mitochondrial membrane potential. Theriogenology 77 1280-1289.

Mendoza N, Casao A, Perez-Pe R, Cebrian-Perez JA & Muino-Blanco T 2013 New insights into the mechanisms of ram sperm protection by seminal plasma proteins. Biology of reproduction 88 149.

Moran JM, Madejon L, Ortega Ferrusola C & Pena FJ 2008 Nitric oxide induces caspase activity in boar spermatozoa. Theriogenology 70 91-96.

Nauc V, De Lamirande E, Leclerc P & Gagnon C 2004 Inhibitors of phosphoinositide 3-kinase, LY294002 and wortmannin, affect sperm capacitation and associated phosphorylation of proteins differently: Ca2+-dependent divergences. Journal of andrology 25 573-585.

Nunez-Martinez I, Moran JM & Pena FJ 2007a Identification of sperm morphometric subpopulations in the canine ejaculate: do they reflect different subpopulations in sperm chromatin integrity? Zygote 15 257-266.

Nunez-Martinez I, Moran JM & Pena FJ 2007b Sperm indexes obtained using computer-assisted morphometry provide a forecast of the freezability of canine sperm. Int J Androl 30 182-189.

Ortega Ferrusola C, Gonzalez Fernandez L, Macias Garcia B, Salazar-Sandoval C, Morillo Rodriguez A, Rodriguez Martinez H, Tapia JA & Pena FJ 2009 Effect of cryopreservation on nitric oxide production by stallion spermatozoa. Biology of reproduction 81 1106-1111.

Ortega Ferrusola C, Gonzalez Fernandez L, Salazar Sandoval C, Macias Garcia B, Rodriguez Martinez H, Tapia JA & Pena FJ 2010 Inhibition of the mitochondrial permeability transition pore reduces "apoptosis like" changes during cryopreservation of stallion spermatozoa. Theriogenology 74 458-465.

Ortega-Ferrusola C, Garcia BM, Gallardo-Bolanos JM, Gonzalez-Fernandez L, Rodriguez-Martinez H, Tapia JA & Pena FJ 2009 Apoptotic markers can be used to forecast the freezeability of stallion spermatozoa. Animal reproduction science 114 393-403.

Ortega-Ferrusola C, Sotillo-Galan Y, Varela-Fernandez E, Gallardo-Bolanos JM, Muriel A, Gonzalez-Fernandez L, Tapia JA & Pena FJ 2008 Detection of "apoptosis-like" changes during the cryopreservation process in equine sperm. Journal of andrology 29 213-221.

Parte PP, Rao P, Redij S, Lobo V, D'Souza SJ, Gajbhiye R & Kulkarni V 2012 Sperm phosphoproteome profiling by ultra performance liquid chromatography followed by data independent analysis (LC-MS(E)) reveals altered proteomic signatures in asthenozoospermia. Journal of proteomics 75 5861-5871.

Pena FJ, Johannisson A, Wallgren M & Rodriguez Martinez H 2003a Antioxidant supplementation in vitro improves boar sperm motility and mitochondrial membrane potential after cryopreservation of different fractions of the ejaculate. Anim Reprod Sci 78 85-98.

Pena FJ, Johannisson A, Wallgren M & Rodriguez-Martinez H 2003b Assessment of fresh and frozen-thawed boar semen using an Annexin-V assay: a new method of evaluating sperm membrane integrity. Theriogenology 60 677-689.

Pena FJ, Nunez-Martinez I & Moran JM 2006 Semen technologies in dog breeding: an update. Reproduction in domestic animals = Zuchthygiene 41 Suppl 2 21-29.

Pena FJ, Ortega-Ferrusola C, Tapia JA, Aparicio IM 2012 How stallion sperm age in vitro? Scenario for preservation technologies Journal of Equine Veterinary Science 32 451-454.

Pena FJ, Saravia F, Garcia-Herreros M, Nunez-martinez I, Tapia JA, Johannisson A, Wallgren M & Rodriguez-Martinez H 2005a Identification of sperm morphometric subpopulations in two different portions of the boar ejaculate and its relation to postthaw quality. Journal of andrology 26 716-723.

Pena FJ, Saravia F, Johannisson A, Walgren M & Rodriguez-Martinez H 2005b A new and simple method to evaluate early membrane changes in frozen-thawed boar spermatozoa. Int J Androl 28 107-114.

Petrunkina AM & Harrison RA 2010 Systematic misestimation of cell subpopulations by flow cytometry: a mathematical analysis. Theriogenology 73 839-847.

Petrunkina AM & Harrison RA 2011a Cytometric solutions in veterinary andrology: Developments, advantages, and limitations. Cytometry. Part A : the journal of the International Society for Analytical Cytology 79 338-348.

Petrunkina AM & Harrison RA 2011b Mathematical analysis of mis-estimation of cell subsets in flow cytometry: viability staining revisited. Journal of immunological methods 368 71-79.

Pujianto DA, Curry BJ & Aitken RJ 2010 Prolactin exerts a prosurvival effect on human spermatozoa via mechanisms that involve the stimulation of Akt phosphorylation and suppression of caspase activation and capacitation. Endocrinology 151 1269-1279.

Qiao Q, Jiang Y & Li G 2013 Inhibition of the PI3K/AKT-NF-kappaB pathway with curcumin enhanced radiation-induced apoptosis in human Burkitt's lymphoma. Journal of pharmacological sciences 121 247-256.

Said TM, Gaglani A & Agarwal A 2010 Implication of apoptosis in sperm cryoinjury. Reproductive biomedicine online 21 456-462.

Sakkas D, Moffatt O, Manicardi GC, Mariethoz E, Tarozzi N & Bizzaro D 2002 Nature of DNA damage in ejaculated human spermatozoa and the possible involvement of apoptosis. Biology of reproduction 66 1061-1067.

Sandilos JK, Chiu YH, Chekeni FB, Armstrong AJ, Walk SF, Ravichandran KS & Bayliss DA 2012 Pannexin 1, an ATP release channel, is activated by caspase cleavage of its pore-associated C-terminal autoinhibitory region. The Journal of biological chemistry 287 11303-11311.

Schulz KR, Danna EA, Krutzik PO & Nolan GP 2007 Single-cell phospho-protein analysis by flow cytometry. Current protocols in immunology / edited by John E. Coligan ... [et al.] Chapter 8 Unit 8 17.

Schulz KR, Danna EA, Krutzik PO & Nolan GP 2012 Single-cell phospho-protein analysis by flow cytometry. Current protocols in immunology / edited by John E. Coligan ... [et al.] Chapter 8 Unit 8 17 11-20.

Sethi G, Ahn KS, Sung B, Kunnumakkara AB, Chaturvedi MM & Aggarwal BB 2008 SH-5, an AKT inhibitor potentiates apoptosis and inhibits invasion through the suppression of anti-apoptotic, proliferative and metastatic gene products regulated by IkappaBalpha kinase activation. Biochemical pharmacology 76 1404-1416.

Shimokawa K, Oshiro R, Yamanaka K, Ashizawa K, Ohta S & Tatemoto H 2012 Improvement of the post-thaw qualities of Okinawan native Agu pig sperm frozen in an extender supplemented with antiapoptotic PTD-FNK protein. Theriogenology 78 1446-1455.

Tazzari PL, Cappellini A, Bortul R, Ricci F, Billi AM, Tabellini G, Conte R & Martelli AM 2002 Flow cytometric detection of total and serine 473 phosphorylated Akt. Journal of cellular biochemistry 86 704-715.

Yang CM, Lee IT, Lin CC, Wang CH, Cherng WJ & Hsiao LD 2013 c-Src-dependent MAPKs/AP-1 activation is involved in TNF-alpha-induced matrix metalloproteinase-9 expression in rat heart-derived H9c2 cells. Biochemical pharmacology 85 1115-1123.

FIGURES

FIGURE 1. Representative cytograms of the simultaneous flow cytometric assessment of subtle

membrane changes, viability and reactive oxygen species (ROS). Samples were stained with Hoechst

33342, YoPro-1, Ethidum Homodimer and CellROX as described in material and methods. Hierarchical

gating was applied to exclude debris from the analysis and simultaneously measure viability, apoptosis,

necrosis and ROS. (A), Hoescht 33342 fluorescence was detected at V1 channel (Ex 405 Band Pass filter

450/50 nm), and a gate was applied to positive (DNA containing particles) events to gate out debris.

Then, the gated region was analyzed (B) YoPro-1 was detected on B1 channel (Ex 488 Band Pass Filter

525/50), ethidium homodimer was detected in B3 (Ex 488 Band pass filter 655-730nm) and (C)

CellROX was detected in R1 (Ex 635 nm Band pass filter 655-730 nm). (D) Positive controls for

oxidative stress (800M plus 200L H202)

FIGURE 2. Representative cytograms of the simultaneous flow cytometric assessment of caspase 3 and 7

and mitochondrial activity in stallion spermatozoa. Samples were stained with CellEvent ,ethidium

homodimer, mitotracker deep red and Hoechst 33342 as described in material and methods. Hierarchical

gating was applied to exclude debris from the analysis and simultaneously measure viability, caspase

activity, necrosis and mitochondrial activity. (A), Hoescht 33342 fluorescence was detected at V1 channel

(Ex 405 Band Pass filter 450/50 nm), and a gate was applied to positive (DNA containing particles)

events to gate out debris. Then, the gated region was analyzed (B) Caspase 3 and 7 were detected on B1

channel (Ex 488 Band Pass Filter 525/50), ethidium homodimer was detected in B3 (Ex 488 Band pass

filter 655-730nm) and (C) Mitotracker deep red was detected in R1 (Ex 635 nm Band pass filter 655-730

nm). (D) Caspase 3 and 7 positive controls (frozen thawed sperm).

FIGURE 3. Subcellular localization of Phospo Akt in stallion spermatozoa. Ejaculated stallion

spermatozoa were washed and stained using specific anti phospho Akt antibodies and visualized under

confocal laser microscopy as described in material and methods. Phosporylated Akt was present in the

post acrosomal region and mid piece. A Transmission image, B Florescence recorded at 540/30 emission

filter, C merged images.

FIGURE 4. Effect of the Akt inhibitor SH5 on Akt phosphorylation at Ser473 and Thr308 of stallion

spermatozoa. Stallion spermatozoa were washed and incubated in presence of Akt inhibitor (0 and 30

M), up to four hours, then were stained with specific antiphospho Akt antibodies (Phospho-Akt (Ser

473) –Alexa Fluor 488 conjugate and Phospho-Akt (Thr308) Alexa fluor 647 conjugate as described in

material and methods. Samples were analyzed flow cytometrically. Events in the lower right quadrant

represent spermatozoa positive for pAkt Ser 473, events in upper right quadrant represent spermatozoa

positive for pAkt Ser 473 and Thr308 simultaneously and event in upper left quadrant represent

spermatozoa positive for pAkt Thr308. The specificity of the antibodies was validated using western

blotting (upper left corner) as described in material and methods, lanes 1 and 2 represent sperm lysates

from two different stallions, lane 3 positive controls (pancreatic acini)

FIGURE 5. Effect of inhibition of Akt phosphorylation on stallion sperm motility and kinematics.

Stallion spermatozoa were washed and resuspended in BWW supplemented with 1% PVA in presence of

Akt inhibitor SH5 at 0, 10, 20 and 30 M for up to 6 hours. A after 1 hour of incubation at 37ºC, B after 2

hours, C after 4 hours and D after 6 hours of incubation at 37ºC. TM %percentage of total motile

spermatozoa, PM % percentage of progressive motile spermatozoa, VCL (m/s) circular velocity, VAP

(m/s) average velocity VSL (m/s) straight line velocity. ** p<0.01, * p<0.05

FIGURE 6. Effect of inhibition of Akt on stallion sperm membrane permeability and integrity. Stallion

spermatozoa were washed and resuspended in BWW supplemented with 1% PVA in presence of Akt

inhibitor SH5 at 0, 10, 20 and 30 M for up to 6 hours. A after 1 hour of incubation at 37ºC, B after 2

hours, C after 4 hours and D after 6 hours of incubation at 37ºC. Intact % percentage of spermatozoa with

completely intact membranes YoPro+ percentage of spermatozoa with increased permeability in their

membranes, YoPro+/Eth+ percentage of apoptotic spermatozoa Eth+ percentage of dead spermatozoa. *

p<0.05

FIGURE 7. Identification and function of the Pannexin-1 channel in stallion spermatozoa. Left panel

western blotting showing the presence of pannexin-1 in stallion sperm lysates, lane -1 control, lanes 2-4

sperm lysates from three different stallions. Right panel whisker and box graphics showing the effect of

two different blockers of the pannexin-1 channel in the uptake of YoPro-1 by stallion spermatozoa. The

uptake of YoPro-1 was significantly inhibited by both inhibitors (* p<0.05)

FIGURE 8. Effect of inhibition of Akt on caspase 3 and 7 activation in stallion spermatozoa. Samples

were washed and resuspended in BWW supplemented with 1% PVA in presence of Akt inhibitor SH5 at

0, 10, 20 and 30 M for up to 6 hours. A after 1 hour of incubation at 37ºC, B after 2 hours, C after 4

hours and D after 6 hours of incubation at 37ºC. Live % percentage of live spermatozoa, casp3+ %

percentage of live spermatozoa with activated caspases 3 and 7, cas+/Eth+% percentage of dead

spermatozoa with activated caspases 3 and 7. ** p<0.01, * p<0.05

FIGURE 9. Effect of two different inhibitors of Akt (Akt ½ kinase inhibitor and SH-5) on motility,

velocity, caspase 3 and 7 activation and Akt phosphorylation on stallion spermatozoa. Samples were

washed and resuspended in BWW media, incubated for 1 hour at 37ºC in presence of 0 or 30 M of Akt

1/2 kinase inhibitor and 30 M of SH-5. A.- Effects in stallion sperm motility, TM % percentage of total

motile sperm, PM % percentage of progressive motile sperm. B.- Effects of stallion sperm velocities,

VCL (m/s) circular velocity, VSL (m/s) straight line velocity, VAP (m/s) average velocity. C.-

Effects on caspase 3 and 7 activation, live percentage of live sperm, Casp+ percentage of live sperm with

active caspases, Casp+/Eth+ percentage of dead sperm with active caspases. D.- Effect on Akt

phosphorylation Thr308 percentage of spermatozoa with Akt phosphorylated at threonine 308, Ser473

percentage of spermatozoa with Akt phosphorylated at serine473, The308+ Ser 473 percentage of

spermatozoa with Akt phosphorylated simultaneously at threonine 308 and serine 473. * p<0.05, **

p<0.01.

FIGURE 10. Effect of inhibition of Akt on the percentage of active mitochondria in stallion spermatozoa

determined after mitotracker staining. Samples were washed and resuspended in BWW supplemented

with 1% PVA in presence of Akt inhibitor SH5 at 0, 10, 20 and 30 M for up to 6 hours A after 1 hour of

incubation at 37ºC B after 2 hours, C after 4 hours and D after 6 hours of incubation at 37ºC. * p<0.05

FIGURE 11. Effect of inhibition of Akt on the percentage of active mitochondria in stallion spermatozoa

determined after JC-1 staining. Samples were washed and resuspended in BWW supplemented with 1%

PVA in presence of Akt inhibitor SH5 at 0, 10, 20 and 30 M for up to 6 hours. A after 1 hour of

incubation at 37ºC B after 2 hours, C after 4 hours and D after 6 hours of incubation at 37ºC. High % of

spermatozoa with high mitochondrial membrane potential, Intermediate % percentage of spermatozoa

depicting simultaneously mitochondria with high and low potential, Low % percentage of spermatozoa

showing low potential mitochondria. ** p<0.01, * p<0.05

FIGURE 12. Effect of inhibition of Akt on the percentage of stallion spermatozoa showing oxidative

stress. Samples were washed and resuspended in BWW supplemented with 1% PVA in presence of Akt

inhibitor SH5 at 0, 10, 20 and 30 M for up to 6 hours A after 1 hour of incubation at 37ºC B after 2

hours, C after 4 hours and D after 6 hours of incubation at 37ºC.